Molecular Mechanism Of Hydroxysafflor Yellow A Regulating AMPK/NLRP3 Pathway Against Myocardial Ischemia Reperfusion Injury

Acute myocardial infarction seriously threatens human health.Early blood flow recovery plays an important role in maintaining cardiac function and reducing myocardial cell injury.However,with the recovery of blood flow,further damage to myocardial tissue structure and heart function is called myocardial ischemia reperfusion injury(MIRI).Inflammatory response is one of the important pathological mechanisms of MIRI.Ischemia/reperfusion(I/R)induces the activation of NLRP3 inflammatory bodies,and the resulting IL-1β,IL-18 and caspase-1 are involved in the MIRI process.Autophagy can inhibit the activation of inflammatory cells and the secretion of IL-1β and IL-18.Autophagy plays an important role in MIRI,and moderate autophagy inhibits the damage after myocardial infarction.Therefore,regulating autophagy to inhibit NLRP3 inflammasome activation is an effective way to reduce MIRI.AMP-activated protein kinase(AMPK)can negatively regulate the inflammatory response mediated by NLRP3 activation through autophagy and other pathways.Hydroxysafflor yellow A(HSYA)is the main flavonoid active ingredient of safflower and safflower yellow for injection.Many pharmacological activities of safflower and safflower yellow for injection are related to HSYA.Safflower is a traditional representative medicine for promoting blood circulation and removing blood stasis,which is widely used in the treatment of cardiovascular and cerebrovascular diseases.Safflower flavonoids are the main effective substances of safflower and have significant protective effects on cardiovascular system.Safflower flavonoids have protective effects on hypoxia/reoxygenation(H/R)-induced myocardial cell injury andthe mechanism of this is related to the inhibition of NLRP3 inflammasome activation.However,the material basis for the protective effect of safflower flavonoids on MIRI remains unclear.Whether HSYA can protect MIRI by inhibiting NLRP3 inflammatory cells and its mechanism remains to be determined.In this study,the H/R model of H9c2 cardiomyocytes was used to investigate the protective effect of five safflower flavonoids on MIRI,and then the cellular and animal models were used to investigate the protective effect of HSYA on MIRI and the molecular mechanism of autophagy inhibition of NLRP3 inflammasome activation.The main research results are as follows:1.Five safflower flavonoids,hydroxysafflor yellow A,dehydrated safflower yellow B,kaempferol,hollywood phenol and apigenin,were prepared at different concentrations,pretreatment for 6 h.MTT method was used to detect the effects of different safflower flavonoids on normal H9c2 cardiomyocytes,and the safe dose range of five safflower flavonoids was determined.Then the five safflower flavonoids were pre-treated for 4 h,12 h and 24 h,and then hypoxia for 6 h and reoxygenation for 12 h.The protective effects of five safflower flavonoids on H/R-induced H9 c2 myocardial cell injury were detected by MTT method.The results showed that hydroxysafflor yellow A and dehydrated safflor yellow B had no significant effect on normal H9c2 cardiomyocytes in the dose range set in this experiment.Apigenin and kaempferol decreased the survival rate of H9c2 cardiomyocytes when the dose reached 25 μM,while hollywood decreased the survival rate of H9c2 cardiomyocytes when the dose was greater than 50 μM.Five safflower flavonoids all had protective effects on H/R-induced H9c2 cardiomyocytes injury.Among them,hydroxysafflor yellow A,dehydrated safflower yellow B and kaempferol showed strong protective effects on H/R-induced H9c2 cardiomyocytes injury.2.Adult SD rats were ligated with left anterior descending branch for 30 min,and then reperfusion for 24 h to establish MIRI model to study the protective effect and mechanism of HSYA on MIRI in rats.TTC staining was used to measure and calculate the myocardial infarction area of rats in different groups.TUNEL kit was used to detect the apoptosis of myocardial tissue.Morphological changes of myocardial tissue were detected by HE staining.The levels of CK-MB,LDH and AST in serum of rats were detected by biochemical kit.The levels of inflammatory factors such as TNF-α,IL-1β and IL-18 in serum of rats in different groups were detected by ELISA kit.The expression of NLRP3 inflammasome and other related proteins in myocardial tissues of rats in different groups were detected by Western blot and immunohistochemistry to evaluate the effect of HSYA on inflammatory injury.Western blot was used to detect the expression of autophagy-related proteins Atg5,BECN1,P62 and LC3B to evaluate the effect of HSYA on autophagy.The effect of HSYA on AMPK/mTOR signaling pathway was detected by Western blot.The results showed that HSYA had a protective effect on MIRI in rats,which could reduce myocardial infarction area and myocardial dysfunction.Compared with ischemia-reperfusion injury group,HSYA could inhibit the expression of mTOR,increase the phosphorylation level of AMPK,inhibit cardiomyocyte apoptosis,reduce the level of serum inflammatory cytokines in rats,induce autophagy,and inhibit the activation of NLRP3 inflammasome.3.To study the protective effect of HSYA on H/R-induced H9c2 cardiomyocytes injury and its mechanism.JC-1 staining was used to observe the changes of mitochondrial membrane potential in cells,flow cytometry was used to detect early apoptosis(AnnexinV/PI double staining),and biochemical kit was used to detect caspase-3 activity to study the protective effect of HSYA on H/R-induced cardiomyocyte apoptosis.The secretion of IL-1β in the supernatant was detected by ELISA kit,the activity of Caspase-1 was detected by biochemical kit,and the expression of NLRP3 inflammasome-related protein was detected by Western blot to evaluate the protective effect of HSYA on inflammatory injury.The stable cell line of H9c2 cells of autophagy double-labeled lentivirus(RFP-GFP-LC3)was constructed,and the autophagy flow was monitored in real time.The expression of autophagy-related proteins such as LC3 was detected by Western blot to evaluate the effect of HSYA on autophagy.The effect of HSYA on protein phosphorylation of AMPK signaling pathway was detected by Western blot.The results showed that HSYA pretreatment had a protective effect on H/R-induced H9c2 myocardial cell injury,improved myocardial cell viability,maintained mitochondrial membrane potential,reduced cardiomyocyte apoptosis,reduced caspase-3 activity,and inhibited the activation of NLRP3 inflammasome in H/R-induced injury.The protective effect of HSYA on H/R-induced myocardial cell injury can be inhibited by Compound C,indicating that HSYA can protect H/R-induced myocardial cell injury by increasing AMPK phosphorylation level,improving autophagy,and negatively regulating NLRP3 inflammasome activation.This paper has proved for the first time that HSYA can improve autophagy and negatively regulate the activation of NLRP3 inflammasome by increasing AMPK phosphorylation level,thereby playing an anti-MIRI role and providing a scientific basis for more accurate clinical application of safflower.It was also clarified that promoting autophagy and inhibiting the activation of NLRP3 inflammatory cells could be used as a new way for drugs to protect MIRI,providing a new idea for the discovery of new drugs against MIRI.